The presence of latent reservoirs has prevented the eradication of Human Immunodeficiency Virus (HIV) from infected patients successfully treated with antiretroviral therapy. HIV latency is poorly understood, partly because of the lack of an in vitro model. Using an HIV molecular clone tagged with green fluorescent protein (GFP), we observed that HIV latency occurred reproducibly with low frequency (1.5%) during infection in vitro. Clonal cell lines derived from this latent population showed no basal expression, but could be induced after treatment with biological and pharmacological agents. Direct sequencing of integration sites demonstrated that HIV frequently integrated in heterochromatin in latent clones in contrast to acute infection where heterochromatin is disfavored as a site for integration. These observations are in agreement with our previous work showing that the chromatin environment at the site of integration of HIV into the genome plays an important role in its transcriptional activity. Here, we propose to further study the role of the HIV integration site environment in latency by conducting an exhaustive analysis of integration sites in latent cells obtained after in vitro infection of a lymphoid cell line or after infection of primary CD4 T cells. We will also sequence sites of integration obtained from HIV infected individuals or from SIV infected macaques. The integration sites will be characterized in terms of posttranslational modifications of chromatin and bound factors to identify a unique signature of modifications associated with latency. We will use a retrovirus-mediated mutagenesis strategy to identify cellular genes that control HIV latency and will use retrovirus-vector mediated expression cloning to characterize cellular genes that mediate the reactivation of HIV latency. Finally, we will expand a screen for small molecules that reactivate latent HIV expression and will attempt to identify the target of action of these molecules using forward genetic approaches. We anticipate that these experiments will increase our understanding of the molecular mechanism of HIV latency and open new therapeutic opportunities for this important problem.